Retinoic acid (RA) exerts profound effects on many cellular processes, including embryonic pattern formation and cellular differentiation. RA can also regulate the growth and differentiation of a variety of pre-malignant and neoplastic cell types. Recent studies have shown that RA exerts anti- tumorigenic and growth inhibitory effects both in mammary carcinoma cell lines, and in a variety of other types of cancers, including squamous carcinoma of the head and neck, and of the cervix. Although the exact mechanisms by which retinoic acid can function as morphogen, a teratogen, and an anti-tumorigenic agent are not understood, a number of genes are regulated by RA during the induction of differentiation and during embryogenesis. One such gene Hoxa-1, is a member of the homeobox gene family. Hoxa-1 was first isolated from a cDNA library made from an RA- treated F9 murine embryonic carcinoma cell line. Expression of the Hoxa-1 gene is up-regulated by RA in a protein synthesis-independent fashion which indicates that the Hoxa-1 gene is a primary target gene of RA. Moreover, an RA-responsive enhancer was identified in the 3' region of the Hoxa-1 gene, and this RA responsive enhancer is identical to that found in the RAR-beta gene. These findings indicate that RA directly regulates the transcription of at least one of the homeobox genes, Hoxa-1. Recently, several groups have reported abnormally high Hoxa-1 expression in both human breast carcinoma cell lines and mouse mammary tumors. It has also been shown that RA can inhibit cell growth only in human breast carcinoma cell lines which possess a functional estrogen receptor. It is not clear how the Hoxa-1 gene is regulated in the breast carcinoma cells, and whether there is any correlation between aberrant Hoxa-1 expression and expression of estrogen receptor among malignant breast epithelial cells. In this proposal, I plan to investigate the regulation of Hoxa-1 gene expression in breast carcinoma cells. Specifically, I will: l) examine the Hoxa-1 gene expression in normal vs. malignant breast epithelial cells, 2) determine the genomic regulatory sequences of the Hoxa-1 gene that are involved in or required for its aberrantly high expression in breast cancer cell lines, 3) define the precise regulatory sequences that are involved in the aberrant expression of the Hoxa-1 gene in the breast cancer cell lines, 4) investigate the regulation of the expression of the Hoxa-1 gene in a transgenic mouse model system for breast tumor development. These experiments should provide important insights into the regulation of the Hoxa-1 gene in breast cancer cells.